CN105807041A - Kit for detecting efficient cyhalothrin residue - Google Patents
Kit for detecting efficient cyhalothrin residue Download PDFInfo
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- CN105807041A CN105807041A CN201610159561.5A CN201610159561A CN105807041A CN 105807041 A CN105807041 A CN 105807041A CN 201610159561 A CN201610159561 A CN 201610159561A CN 105807041 A CN105807041 A CN 105807041A
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Abstract
The invention provides a preparing and using method of an enzyme linked immunosorbent assay kit for detecting a efficient cyhalothrin residue. The kit is composed of an elisa plate coated with an efficient cyhalothrin coupling antigen, an efficient cyhalothrin standard sample, an enzyme-labelled second antibody, an antibody concentrated solution, a substrate solution, a stop solution, a concentrated washing solution and a concentrated compounded solution. The efficient cyhalothrin coupling antigen is obtained by coupling efficient cyhalothrin hapten and carrier protein, and the carrier protein can be bovine serum albumin, egg albumin, hemocyanin, thyroprotein and human serum albumin; the coupling efficient cyhalothrin hapten is prepared through reaction of efficient cyhalothrin and succinic anhydride. The kit has the advantages of being fast in detection, accurate in measurement result, low in detection cost and high in sensitivity and can play an important role in detecting and monitoring the efficient cyhalothrin residue on site.
Description
Technical field
The invention belongs to enzyme linked immunosorbent detection field, be specifically related to preparation and the application of a kind of gamma cyhalothrin test kit, it is particularly well-suited to the detection of gamma cyhalothrin residual.
Background technology
Gamma cyhalothrin, English name Lambda-Cyhalothrin, it is also called Ai Kening, λ-Grenade (ICI)., belong to pyrethroid, there is the hygienic insecticide of action of contace poison, chemical name: alpha-cyano-3-benzyloxy phenoxy base-3-(2-chloro-3,3, the fluoro-1-acrylic of 3-trichlorine)-2,2-dimethyl cyclopropane carboxylic acid's ester (Z)-(1R, 3R), S-ester and (Z), (1S, 3S), the 1:1 mixture of R-ester.There is action of contace poison, be that the extremely effective one of public health insect is extensively imitated insecticide, have and knock down the advantages such as speed is fast, it is strong to knock down power, and dosage is few.Pathophorous vector pests can be eliminated and prevent and treat various sanitary insect pests.Pyrethroid is known as a new breakthrough of pesticide, is historical 3rd milestone of insecticide.In agricultural production, of many uses.
Detection to gamma cyhalothrin residual, country has formulated strict residue detection standard, such as GB/T5009-2008 " in vegetable food the qualification of organochlorine and the multiple residual of pyrethroid pesticide ", GB/T19649-2006 " in Cereals the mensuration of 475 kinds of pesticide and the flat residual quantity of related chemistry ", SN/T1117-2008 " in import and export food multiple Determining pyrethroid pesticide residues assay method gas chromatography ", existing method for detecting residue is gas chromatography and high performance liquid chromatography, detection method depends on large-scale instrument and professional technique testing staff, the detection time is long, detection process needs use organic solvent, environmental pollution can be caused.Seek one quickly, effectively, detection method and means accurately, imperative.Enzyme-linked immune detection method has the advantages that sensitivity is good, degree of accuracy is high, testing cost is low, simple to operate, the detection time is short, can realize that multisample detects simultaneously, plays an important role in food safety quickly detects.
At present, some achievements there are is to report the test kit of pyrethroid both at home and abroad, patent of invention " indirect competitive enzyme-linked immunosorbent assay kit of cyhalothrin residual " (patent No.: 200810101544.1) has been applied for as China Agricultural University is permitted ship et al., with hydroxy phenylpropionic acid for raw material, through six-step process, acquisition hapten 3-[(±)-(cis)-3-[(Z)-3-chloro-4, 4, the fluoro-1-ethyl-2 of 4-tri-, 2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid, with cyhalothrin polyclonal antibody for raw material, set up the enzyme-linked immunosorbent assay kit of detection cyhalothrin residual, it is loaded down with trivial details to there is hapten reactions steps in this technical scheme, subsequent purification workload is big, and the inferior position that polyclonal antibody is good not as monoclonal antibody specificity.Carry out the enzyme linked immunological kit detected currently for gamma cyhalothrin residual, do not see relevant report temporarily.
Summary of the invention
The present invention intends providing a kind of test kit being specifically designed for gamma cyhalothrin residue detection, adopt indirect competitive ELISA method, and a kind of efficient, accurate, simplicity, the qualitative and quantitative analysis method being suitable to batch samples screening and test kit preparation method are provided, the detection of Nicotiana tabacum L. is limited to 900 μ g/kg by the method.
Test kit of the present invention, it includes: the multiple solution composition of the ELISA Plate of gamma cyhalothrin coupled antigen, gamma cyhalothrin standard substance, ELIAS secondary antibody, specific antibody, substrate solution, stop buffer, concentrated cleaning solution and concentration, described ELIAS secondary antibody is enzyme labelling anti antibody.
Test kit of the present invention, based on technical method prepared by gamma cyhalothrin hapten, the method is with gamma cyhalothrin for raw material, through single step reaction, can obtaining hapten, it is simple that synthetic method has technique, the feature that response rate is high, and remain the chemical constitution of gamma cyhalothrin to greatest extent, molecular structural formula is shown in Formulas I:
(Formulas I).
The preparation method of gamma cyhalothrin hapten compound, comprises the following steps:
1) with gamma cyhalothrin for raw material, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2) in solution I, succinic anhydrides, stirring and dissolving under room temperature are added；
3) add aluminum chloride solid and carry out catalytic reaction；
4) TLC lamellae monitoring reaction carries out, until not having raw material or raw material point very shallow, and stopped reaction；
5) silicagel column purifies, and concentration obtains product, is hapten.
In above-mentioned reaction, reaction temperature is room temperature, and reaction pressure is normal pressure, and the reaction mol ratio of gamma cyhalothrin and succinic anhydrides is 1:(1-3), catalyst and gamma cyhalothrin mass ratio are 1%-5%.
The gamma cyhalothrin hapten synthesis route of the present invention is as follows:
In the present invention, the gamma cyhalothrin used, its chemistry is by name: alpha-cyano-3-benzyloxy phenoxy base-3-(2-chloro-3,3, the fluoro-1-acrylic of 3-trichlorine)-2,2-dimethyl cyclopropane carboxylic acid's ester (Z)-(1R, 3R), S-ester and (Z), (1S, 3S), the 1:1 mixture of R-ester, molecular formula is: C23H19ClF3NO3。
The method adopting the present invention can prepare the hapten compound that structural formula is I, and this hapten remains the chemical constitution of gamma cyhalothrin to greatest extent, and introduces active group-OH, adds junction point for antigen preparation.
This chemical reaction adopts one-step method to complete, and yield is up to 51.6%
The preparation method that the present invention also provides for a kind of gamma cyhalothrin antigen.
Weigh a certain amount of hapten and be dissolved in DMF solution, add EDC and NHS(to be dissolved in the water) carry out activation 30min, join carrier protein (being dissolved in 5mL containing in 30%DMF aqueous solution) and carry out coupling, prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, prepares antibody for animal immune after having dialysed.Structural formula is Formula II:
(Formula II).
Carrier protein can be bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumin.
Described gamma cyhalothrin specific antibody is to prepare using gamma cyhalothrin coupled antigen as immunogen, described gamma cyhalothrin specific antibody can be gamma cyhalothrin monoclonal antibody or gamma cyhalothrin polyclonal antibody, wherein preferred gamma cyhalothrin monoclonal antibody.
The marker enzyme of described ELIAS secondary antibody is horseradish peroxidase or antibacterial extraction alkaline phosphatase, wherein preferred horseradish peroxidase；Marker enzyme and anti antibody are carried out coupling and obtain by the anti antibody employing glutaraldehyde method of enzyme labelling or Over-voltage protection.
For more convenient on-site supervision and great amount of samples examination, described test kit also includes gamma cyhalothrin standard solution, substrate solution, stop buffer, cleaning mixture, redissolution liquid.
Described standard solution 6 bottles, concentration respectively 0 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L, 2430 μ g/L.
When marker enzyme is horseradish peroxidase, described nitrite ion is made up of substrate nitrite ion A liquid and substrate nitrite ion B liquid, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is sulphuric acid or the hydrochloride buffer of 1-2mol/L；When marker enzyme is alkaline phosphatase, described nitrite ion is to nitro phosphate buffer, and described stop buffer is 1-2mol/L sodium hydroxide solution.
It is 7.2-7.5 that described cleaning mixture is preferably pH value, containing 0.8%-1.0% tween 20,0.03 ‰-0.05 ‰ sodium azide preservatives, 0.1-0.3mol/L phosphate buffer, described percentage ratio is percent weight in volume.
Described redissolution liquid be preferably pH value be 7.0, the phosphate buffer of 0.02mol/L, described percentage ratio is percent weight in volume.
Wherein in ELISA Plate preparation process, the used buffer that is coated be pH value is 9.2-9.6, the carbonate buffer solution of 0.1-0.2mol/L, confining liquid is pH value is 7.1-7.5, and containing the phosphate buffer of 1%-3% casein, 0.1-0.3mol/L, described percentage ratio is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: with being coated buffer, coating antigen is diluted to 30 μ g/ml, every hole adds 100 μ l, 37 DEG C of lucifuges hatch 2h or 4 DEG C overnight, and liquid in hole of inclining washs 1 time with cleaning mixture, stop 30s, patting dry, then add 150 μ l confining liquids in every hole, 37 DEG C of lucifuges hatch 1-2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves.
The Cleaning Principle of the present invention is:
Pre-coated gamma cyhalothrin coupled antigen in ELISA Plate, after adding sample solution or standard solution, add gamma cyhalothrin specific antibody solution, the gamma cyhalothrin medicine of residual and coated gamma cyhalothrin coupled antigen competition gamma cyhalothrin specific antibody in ELISA Plate in sample, add ELIAS secondary antibody and be amplified effect, develop the color with nitrite ion, the content of sample absorbance and gamma cyhalothrin medicine is negative correlation, compares with standard curve and can obtain the residual quantity of gamma cyhalothrin in sample；Simultaneously according to the depth of color in ELISA Plate, the comparison with the gamma cyhalothrin standard solution color of series concentration can the concentration range of gamma cyhalothrin residual quantity in judgement sample roughly.
The present invention detects the enzyme linked immunological kit of gamma cyhalothrin and mainly adopts competitive ELISA method qualitative or the content of gamma cyhalothrin in detection by quantitative sample；Requiring low to the pre-treatment of sample, sample pretreatment process is simple, can quickly detect batch samples simultaneously；Main agents provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.The enzyme linked immunological kit of the present invention, simple in construction, easy to use, low price, carrying convenience, detection method efficiently, accurately, easy, be suitable to the qualitative, quantitative of batch samples screening.
Accompanying drawing explanation
Fig. 1 gamma cyhalothrin hapten structure schema I.
Fig. 2 gamma cyhalothrin antigenic structure figure Formula II.
Fig. 3 gamma cyhalothrin hapten synthesis reaction scheme.
Fig. 4 gamma cyhalothrin hapten identifies figure (hydrogen spectrogram).
Fig. 5 gamma cyhalothrin hapten identifies figure (carbon spectrogram).
Fig. 6 gamma cyhalothrin ELISA kit standard curve.
Detailed description of the invention
The present invention is expanded on further, it should be appreciated that these embodiments are merely to illustrate the present invention, rather than are used for limiting the scope of the present invention below in conjunction with specific embodiment.
Embodiment one: the preparation of gamma cyhalothrin reagent constituents
One, gamma cyhalothrin hapten synthesis and qualification
Weigh 0.42g(1mmol respectively) gamma cyhalothrin, 0.10g(1mmol) succinic anhydrides and 0.05g aluminum chloride.
1) by 0.42g gamma cyhalothrin, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2) in solution I, 0.10g succinic anhydrides, stirring and dissolving under room temperature are added；
3) add 0.01g aluminum chloride solid and carry out catalytic reaction；
4) TLC lamellae monitoring reaction carries out, until not having raw material or raw material point very shallow, and stopped reaction；
5) silicagel column purifies, and concentration obtains product, is hapten, obtains product 0.205g, yield 48.8%, and reaction technology route is as follows:
Two, gamma cyhalothrin hapten is identified
Take above-mentioned Fig. 1 compound, structural analysis is carried out through proton nmr spectra, as shown in Figure 4, in discovery collection of illustrative plates, the peak near 11ppm (mg/L) is the H on hydroxyl, meanwhile, structural analysis is carried out through carbon-13 nmr spectra, as shown in Figure 5, in carbon spectrogram, the peak near 177ppm is the C on carboxyl, and the success of gamma cyhalothrin hapten synthesis is described.
Three, the preparation of gamma cyhalothrin antigen
1. immunogenic synthesis
Weigh 27.5mg hapten and be dissolved in 4mLDMF solution, add each 60mgEDC and 60mgNHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 110-264mg carrier protein BSA(and be dissolved in 5mL containing in 30%DMF aqueous solution) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, to remove unreacted small-molecule substance, after having dialysed, the immunogen subpackage that will obtain, saves backup in-20 DEG C.
2. the synthesis of coating antigen
Weigh 27.5mg hapten and be dissolved in 4mLDMF solution, add each 60mgEDC and 60mgNHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 100-240mg carrier protein OVA(and be dissolved in 5mL containing in 30%DMF aqueous solution) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, to remove unreacted small-molecule substance, after having dialysed, the immunogen subpackage that will obtain, saves backup in-20 DEG C.
3. the qualification of gamma cyhalothrin antigen
By carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate pH7.4 PB be made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PB, it is scanned within the scope of wavelength 260 ~ 750nm with ultraviolet spectrophotometer, draw the absorption curve of carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate, and calculate its combination ratio.Result shows, there is different absorption curves in three, proving the success of gamma cyhalothrin hapten and carrier protein couplet, the combination of gamma cyhalothrin hapten and bovine serum albumin ratio is for 15-21:1, and the combination of gamma cyhalothrin hapten and oralbumin ratio is for 20-25:1.Molecular structural formula is such as shown in Formula II:
(Formula II).
Four, gamma cyhalothrin monoclonal antibody
1. gamma cyhalothrin monoclonal antibody preparation
Animal immune: gamma cyhalothrin hapten and carrier protein couplet thing immunity 8-10 week old Balb/c mice.
Cell fusion and cloning: take the mice spleen cell after immunity, merge under the effect of fusion agent Polyethylene Glycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
The monoclonal hybridoma strain of gamma cyhalothrin is obtained through screening.The monoclonal hybridoma strain of gamma cyhalothrin can an unbounded quantity of generation gamma cyhalothrin specific antibody, this antibody specificity is for gamma cyhalothrin, and sensitivity reaches 1 μ g/L.
Cell cryopreservation and recovery: hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
2. the mensuration of antibody titer
The titer measuring antibody with indirect competitive ELISA method is 1: 100000～150000.
Indirect competitive ELISA method: with gamma cyhalothrin hapten-bovine serum albumin conjugate coated elisa plate, add gamma cyhalothrin standard substance working solution solution, monoclonal antibody working solution and ELIAS secondary antibody, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3～5 times with PBST cleaning mixture, pat dry with absorbent paper；Add substrate nitrite ion, after 25 DEG C of reaction 15min, add stop buffer and terminate reaction；Set microplate reader and measure every hole absorbance in wavelength 450nm place.
3. the specificity of monoclonal antibody
Antibody specificity refers to ability and the comparison with such antigen-analogues ability of its homospecificity antigen combination, and conventional cross reacting rate is as evaluation criterion.Cross reaction is more little, and the specificity of antibody is then more high.
Gamma cyhalothrin, cypermethrin and 3 kinds of medicines of fenvalerate are done serial dilution by this experiment, carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then it is calculated as follows cross reacting rate:
Result display cross reacting rate is: gamma cyhalothrin 100%, cypermethrin < 50%, fenvalerate < 10%.Gamma cyhalothrin is had stronger specificity and affinity by antibody of the present invention.
Five, the preparation of sheep anti mouse anti antibody
With sheep for immune animal, with Mus source antibody for immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse anti antibody.
Six, the preparation of ELIAS secondary antibody
Over-voltage protection after sheep anti mouse anti antibody is adopted improvement with horseradish peroxidase (HRP) carries out coupling.In reaction system after improvement, enzyme is 1.8-2.4:1 with the molar concentration rate of antibody.
Seven, the preparation of ELISA Plate
With being coated buffer, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 DEG C of lucifuges hatch 2h, liquid in hole of inclining, and wash 1 time with cleaning mixture, stop 30s, patting dry, then add 150 μ l confining liquids in every hole, 37 DEG C of lucifuges hatch 2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves.
Embodiment two: set up the enzyme linked immunological kit of gamma cyhalothrin monoclonal antibody preparation
One, gamma cyhalothrin enzyme linked immunological kit, comprises following each component:
(1) ELISA Plate of gamma cyhalothrin coupled antigen it is coated with.
(2) enzyme labelling anti antibody: horseradish peroxidase-sheep anti mouse anti antibody.
(3) gamma cyhalothrin monoclonal antibody working solution.
(4) standard solution: adopt gradient dilution method preparation standard solution, obtain serial standards 6 bottles, concentration respectively 0.0 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L, 2430 μ g/L, and high standard product 1g/L.
(5) substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
(6) stop buffer is the sulfuric acid solution of 2mol/L.
(7) concentrated cleaning solution is the PBS of 0.3-0.6mol/LpH7.4 of sodium azide preservatives of 0.6%-1.0% tween 20 and 0.01%～0.02%.
(8) concentration redissolution liquid is pH value is 7.4, containing the PBS of 9%-12% oralbumin, 0.1-0.3mol/L.
The main agents of this test kit provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.
The application of embodiment three enzyme linked immunological kit detection actual sample
For Nicotiana tabacum L., utilize test kit that gamma cyhalothrin residual quantity is detected.
1. the pre-treatment of sample
Fresh tobacco leaves Sample pretreatment method:
(1) before detection, sample is shredded into the fragment less than 1cm；
(2) weigh 1.0 ± 0.05g sample minced, put into 5ml methanol and be all saturated with to liquid level；
(3) cover lid, vibrate 1min；
(4) pipette 0.1ml supernatant after standing and join mixing in 900ul0.1MPBS.
Dry tobacco leaf Sample pretreatment method:
(1) before detection, sample is pulverized；
(2) weigh 0.5 ± 0.05g sample pulverized, add 5ml methanol；
(3) cover lid, vibrate 1min；
(4) pipette 0.1ml supernatant after standing and join mixing in 900ul0.1MPBS.
2, detect with test kit
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, 2-8 DEG C of preservation.
(3) numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
(4) adding standard substance/sample 50ml in corresponding micropore, be subsequently adding the mixed liquor 50ml/ hole of antibody concentrated solution and ELIAS secondary antibody working solution, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
(5) plate is washed: carefully open cover plate film, liquid in hole is dried, add wash operating solution (being diluted by 20 × concentrated cleaning solution with deionized water) 250ml/ hole by 1:19 volume ratio, fully washing 4-5 time, every minor tick 10s, sprinkle cleaning mixture in board falling hole, pat dry (bubble not being eliminated after patting dry can be poked with original rifle head) with absorbent paper.
(6) colour developing: add substrate solution A liquid 50ml/ hole, adds substrate solution B liquid 50ml/ hole, and mixing of vibrating gently, with reacting 15min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
(7) measure: add stop buffer 50ml/ hole, mixing of vibrating gently, set microplate reader in 450nm place (suggestion dual wavelength 450/630nm detection, please run through data in 5min), measure every hole OD value.(if without microplate reader, then being not added with stop buffer ocular estimate can judge).
3, Analysis of test results
The percentage absorptance of standard substance or sample is equal to the meansigma methods (diplopore) absorbance divided by first standard (0 standard) of the absorbance of standard substance or sample, then is multiplied by 100%, namely obtains percentage absorptance.With standard substance percentage absorptance for vertical coordinate, with the logarithm of gamma cyhalothrin standard concentration for abscissa, drawing standard curve chart (such as Fig. 6).The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the extension rate of its correspondence and be in sample gamma cyhalothrin actual concentrations.
The mean absorbance values of B standard substance or sample solution
B0The mean absorbance values of 0ppb standard solution
Three, the determination of enzyme linked immunological kit technical parameter
1. test kit sensitivity determination
50% inhibition concentration (i.e. IC50, the drug level corresponding to 50% place of the absorbance of nulling standard solution) and frequently as the sensitivity index evaluating competitive enzyme-linked immune test kit.Utilizing gamma cyhalothrin standard solution to react, according to experimental result drawing standard curve, test kit gamma cyhalothrin standard curve is between 30～2430 μ g/L as seen from the figure.In tobacco sample, gamma cyhalothrin sensitivity is 30 μ g/L.
2. test kit lowest detectable limit
20 parts of blank samples are detected, the concentration corresponding to each percentage light absorption value is found from standard curve, representing detection limit with the meansigma methods of the gamma cyhalothrin measured value of 20 parts of samples plus 3 times of standard deviations, result obtains the method and detects in tobacco sample and be limited to 900 μ g/kg.
3. test kit accuracy and precision
810 μ g/L and the gamma cyhalothrin of 1620 μ g/L concentration is added respectively in blank Nicotiana tabacum L., it is measured, take the test kit of 3 different batches, and each batch is extracted 2 test kits and detects, every kind of sample do 6 times parallel, each sample repeats 2 times, and it is equal 95% ± 25% that result obtains the method response rate to gamma cyhalothrin, variation within batch coefficient <
10%, interassay coefficient of variation < 10%.
4. storage life experiment
Test kit preservation condition is 2-8 DEG C, measures through 12 months, the maximum absorbance value of test kit, IC50Value, gamma cyhalothrin add practical measurement value all within normal range.Doing accelerated ageing and refrigeration test simultaneously, be placed on by test kit in 37 DEG C ,-20 DEG C 6 days, measurement result also indicates that the indices of test kit is normal.Obtain gamma cyhalothrin test kit from the result above to preserve 12 months at 2-8 DEG C.
Claims (3)
1. the test kit detecting gamma cyhalothrin residual, including being coated with the multiple solution composition of the ELISA Plate of gamma cyhalothrin coupled antigen, gamma cyhalothrin standard substance, ELIAS secondary antibody, antibody concentrated solution, substrate solution, stop buffer, concentrated cleaning solution and concentration, described gamma cyhalothrin coupled antigen is to be obtained by gamma cyhalothrin hapten and carrier protein couplet, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin；Gamma cyhalothrin hapten by gamma cyhalothrin and succinic anhydrides react prepare, its molecular structural formula shown in formula I,
(Formulas I).
2. enzyme linked immunological kit according to claim 1, it is characterised in that the concentration of described gamma cyhalothrin standard solution respectively 0 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L, 2430 μ g/L.
3. the method detecting gamma cyhalothrin, comprises the following steps:
(1) sample pre-treatments；
(2) detect with enzyme linked immunological kit；
(3) Analysis of test results.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
| CN113024415A (en) * | 2021-02-05 | 2021-06-25 | 北京勤邦生物技术有限公司 | Cyhalothrin hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN115093347A (en) * | 2022-06-20 | 2022-09-23 | 云南省烟草质量监督检测站 | Hapten for detecting cypermethrin content and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
| CN111812316B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
| CN113024415A (en) * | 2021-02-05 | 2021-06-25 | 北京勤邦生物技术有限公司 | Cyhalothrin hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN115093347A (en) * | 2022-06-20 | 2022-09-23 | 云南省烟草质量监督检测站 | Hapten for detecting cypermethrin content and application thereof |
| CN115093347B (en) * | 2022-06-20 | 2023-09-12 | 云南省烟草质量监督检测站 | Hapten for detecting cypermethrin content and application thereof |
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